T7 promoters have been used to achieve control of the expression of exogenous genes in E. coli, since their expression can be turned on by controlling the presence and expression of the T7 gene 1 (SEQ ID NO: 1) for T7 RNA polymerase (SEQ ID NO: 2) (Studier & Moffat, 1986; Studier et al., 1990; Tabor & Richardson, 1985). Phage T7 RNA polymerase does not recognize E. coli promoters, and vice versa (i.e., E. coli RNA polymerase does not recognize T7 promoters, except for the special “E. coli” one that transcribes gene 1).
The E. coli lac operon has been characterized (see e.g., Dickson et al, 1974; Schultz, Shields, & Steitz, 1991; Oehler, et al. 1990; Flashner & Gralla, 1988). The polycistronic lac operon mRNA molecule encodes three genes: Lac Z, Lac Y and Lac A. The product of the Lac Z coding region functions as a β-galactosidase; this function is required for the metabolism of lactose into glucose and galactose. The product of the Lac Y coding region functions as a lactose permease, which is a membrane-bound transport protein that allows lactose to enter the cell. Lac A is a β-galactosidase transacetylase, and does not appear to be strictly required for lactose metabolism.
The lactose operon genetic control region contains binding sites for 3 control proteins: CAP, RNA polymerase, and lac repressor (Dickson et al, 1974). Mutation L8 (a.k.a. L37) is a G to A transition (Dickson et al., 1977) in the CAP site, which eliminates the possibility of binding or activation by CAP protein when glucose is absent. Since binding by CAP protein activates the lac promoter 16-fold by introducing a 90° bend (Schultz, Shields, & Steitz, 1991), the L8 mutation results in a decreased level of transcription (e.g., to 6%). The phenotype is slightly Lac+ but melibiose negative at 42° C. (Ippen et al. 1968). During genetic analysis of the lac operon, second-site Lac+ (i.e., raffinose+) revertants of this CAP site mutant were selected after UV mutagenesis (Arditti et al, 1968; Silverstone et al. 1970), with the UV5 mutant consisting of a further change of two adjacent base pairs in the RNA polymerase binding site. The resulting promoter region, with a total of 3 base-pair changes, produced a promoter that is stronger than lac wild-type and oblivious to glucose repression, yet is still under control of lac repressor. The resultant L8-UV5 has been widely useful as a model promoter for basic transcription studies. As for the repressor, it is a tetrameric dimer-of-dimers and must bind at least two operators to achieve its full level of repression: Part of the binding of, and control by, the lac repressor depends on operator O2, some 400 bp into the lacZ gene (Oehler, et al. 1990; Flashner & Gralla, 1988). Operator O3, in the I gene, overlaps the CAP site so nearly that it has been proposed to ‘repress’ by interfering with CAP binding (Oehler et al, 1990); in parallel, it may serve to auto-repress the I gene.
Studier (2005) has recently formulated a mixture of sugars consisting of 0.5% glycerol, 0.05% glucose, and 0.2% lactose (ZYM-5052; herein “5052”) to replace the manual addition of inducer IPTG, achieving effective and convenient “auto-induction” of cultures for the purpose of exogenous protein production in E. coli. The host strain for this system is BL21(DE3), a lysogen of phage lambda DE3 carrying the T7 gene 1 (SEQ ID NO: 1) under the control of the lac L8-UV5 promoter.
Auto-induction polypeptide expression systems rely on the principle that an inducer can induce production of target protein but is prevented from doing so by compounds that can be depleted during growth. This allows use of media in which target protein is produced automatically, without the need to monitor growth and add inducer at the proper time. Ideal auto-induction systems allow the host strain to grow in auto-inducing medium without expressing target protein until rather high density, when depletion of inhibitory factors would allow the inducer present in the medium to induce expression, thus producing high concentrations of target protein. For example, glucose in the medium can prevent the uptake and utilization of lactose inducer but when glucose is depleted, the lactose inducer can effectively induce expression of the target protein. Auto-induction is generally preferred over IPTG induction for increased simplicity (e.g., no need to follow culture growth or add inducer at the proper time), increased culture density, and increased concentration of target protein per volume of culture.
But, like all known inducible promoter systems, auto-induction polypeptide expression systems have a residual level of activity or “leakiness”, which leads to the inappropriate transcription and expression of the gene being cloned under the control of the promoter.
The auto-inducing expression system of Studier (2005) is widely used in the industry. Additionally, there exist several modifications to the system. For example, some researchers have inserted the T7 RNA polymerase (gene I) into lacZ (e.g., New England Biolabs), which is reported to provide better control but at the expense of auto-induction (because auto-induction requires lacZ (beta-galactosidase) to make the inducer allolactose). As another example, some researchers have aligned T7 RNA polymerase gene I with the arabinose promoter (e.g., Invitrogen), but one must include arabinose in addition to 5052 sugar mixture for auto-induction. The inventor has observed that the auto-induced level is not as high with this strain, and it has a leaky background in Studier's recommended non-inducing medium. As another example, strains have been selected as resistant to several families of toxic proteins (e.g., Miroux & Walker, 1996; Lucigen). As another example, the gene for T7 lysozyme, a natural inhibitor of T7 RNA polymerase, has been included on another (chloramphenicol-resistant) plasmid (Studier 1991). Further examples include plasmid copy-number control for the T7 expression vector (e.g., pETcoco, Novagen), and the addition of another lac operator (although it now has O1 and O3) to the pET vector series.
Thus there exists a need for improvements to the auto-induction expression system in which the T7 gene 1 is under control of the lac L8-UV5 promoter, especially with regard to reducing the uninduced leaky level of expression.